Smallpox was one of the most important causes of morbidity and mortality worldwide through the first half of the 20th Century. Smallpox is a potential agent of bioterrorism; it has been designated as Category A Priority Pathogen. Despite the declaration of smallpox eradication in 1980, the existence of variola stockpiles and the threat of bioterrorism demand that immunity to smallpox through vaccination be maintained. In recent years, mosquito-borne infections are reemerging or emerging worldwide at an alarming rate. Japanese Encephalitis is a potential agent of bioterrorism; it has been designated as Category C Priority Pathogen. There are no licensed antiviral drugs against JE and only experimental treatments exist for smallpox. Therefore, vaccination and vector control are the only effective preventive tools, with vaccination being the most cost-effective strategy. Because of this effective protection of people requires immediate application of the countermeasures against these pathogens. Therefore, local storage of the countermeasures is preferable to ensure the greatest efficiency in combating the pathogens and to rapidly stop their spread. The best scenario could be achieved if local health professionals and emergency personal had convenient locally available supplies of vaccines that obviate the need for cold to ensure stability during storage. In the past few years, one of our two collaborating groups, the Group of Dr. Thomas Monath at Acambis has developed a new vaccine against Smallpox (Modified Vaccinia Ankara (MVA) and Japanese Encephalitis (ChimeriVax-JE live, attenuated chimeric vaccine). Both products are in clinical development under Food and Drug Administration (FDA) approved INDs. Both are live, attenuated vaccines and as such are susceptible to thermal degradation; hence, the vaccines must be kept refrigerated. During the past few years, the second of our two collaborating groups, Universal Stabilization Technologies (LIST), under the direction of Dr. Victor Bronshtein, has developed exciting new technologies for stabilizing bacteria and other live products. These technologies eliminate the damaging effects of conventional freeze-drying and allow the preparation of a live product that is stable at ambient temperature, eliminating the need for a cold chain. The objective of this grant proposal is to apply the novel technologies for stabilization developed at LIST to the manufacture of micronized ambient temperature stable MVA smallpox vaccine and ChimeriVax JE vaccine. Such formulated vaccines potentially could be delivered without need for reconstitution using transdermal, oral, nasal or inhalation delivery routes. During the first year our target will be vaccines that maintain high viability and potency after drying and subsequent storage at ambient temperatures. Working in collaboration, the Acambis and LIST will culture the vaccines, explore various ambient temperature stabilization approaches, confirm the stability and potency of the preserved vaccine, and test the immunogenicity and efficacy of the preserved vaccine using the mouse model. [unreadable] [unreadable] [unreadable]